The field of this invention is polypeptides involved in cell guidance.
The semaphorins constitute a large family of evolutionally conserved glycoproteins that are defined by a characteristic semaphorin domain of approximately 500 amino acids (1-3). The first vertebrate semaphorin, collapsin-1 in chick, was identified by its ability to induce growth cone collapse (4). Consistent with this function, its mammalian homologue, sema III, has been shown to repel specific subsets of sensory axons (5). As a result of these and other studies, Coll-1/sema III/D has been implicated in the patterning of sensory axon projections into the ventral spinal cord and cranial nerve projections into the periphery (6-11).
Several other semaphorins have also been implicated as repulsive and/or attractive cues in axon guidance, axon fasciculation, and synapse formation (1, 12-17). In addition, members of semaphorin family have been implicated in functions outside the nervous system, including bone skeleton and heart formation (9), immune function (18, 19), tumor suppression (20-22), and conferring drug resistance to cells (23).
Recent studies have identified the first semaphorin receptor as a member of the neuropilin family. Neuropilin-1 is a high affinity receptor for sema III, E and IV, whereas neuropilin-2 binds differentially to the subfamily of secreted semaphorins (24-27).
The vertebrate semaphorin family can be classified into several phylogenetically distinct subfamilies (15). Each subfamily has a unique structural arrangement of protein domains. The secreted members of the semaphorin family contain a characteristic semaphorin domain at the N-terminus, followed by an immunoglobulin (Ig) domain and a stretch of basic amino acids in the carboxyl-terminal region. Between the N-terminal semaphorin domain and the transmembrane spanning region, the transmembrane semaphorins contain several alternative structural motifs including either an Ig domain, a stretch of thrombospondin repeats, or a sequence with no obvious domain homology. Interestingly, semaphorin-like sequences have been identified in the genomes of poxviruses (1) and alcelaphine herpesvirus-1 (28), occupying unique branches of the semaphorin phylogenetic tree. Here we report the identification of a GPI-linked human semaphorinxe2x80x94semaphorin K1xe2x80x94which is homologous to the semaphorin encoded by alcelaphine herpesvirus-1 and show that semaphrin K1 polypeptides and nucleic acids are bioactive in modulating nervous and immune system function.
The invention provides methods and compositions relating to semaphorin K1 (sema K1) polypeptides, related nucleic acids, polypeptide domains thereof having sema K1-specific structure and activity and modulators of sema K1 function. The polypeptides may be produced recombinantly from transformed host cells from the subject sema K1 polypeptide encoding nucleic acids or purified from mammalian cells. The invention provides isolated sema K1 gene hybridization probes and primers capable of specifically hybridizing with the disclosed sema K1-encoding genes, sema K1-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e.g. nucleic acid hybridization screens for sema K1 transcripts), modulating cellular physiology (e.g. by contacting with exogenous sema K1) and in the biopharmaceutical industry (e.g. as immunogens, reagents for isolating other semaphorins, reagents for screening chemical libraries for lead pharmacological agents, etc.).
The nucleotide sequence of a natural cDNA encoding a human sema K1 polypeptide is shown as SEQ ID NO:1, and the full conceptual translate is shown as SEQ ID NO:2. The sema K1 polypeptides of the invention include one or more functional domains of SEQ ID NO:2, which domains comprise at least one of (a) SEQ ID NO:2, (b) at least 100 contiguous residues of SEQ ID NO:2, (c) at least 60 contiguous residues of SEQ ID NO:2, residues 340-634, and (d) at least 12 contiguous residues of SEQ ID NO:2, residues 481-634. A cDNA encoding an alcelaphine herpesvirus semaphorin having sequence similarity to the subject sema K1 polypeptides, and its translate are shown as SEQ ID NO:3 and 4, respectively. Sema K1 specific polynucleotides and polypeptides having human sema K1-specific sequences are readily discernable from alignments of the sequences. Preferred sema K1 polypeptides have one or more human sema K1-specific activities, such as cell surface receptor binding and/or binding inhibitory activity and sema K1-specific immunogenicity and/or antigenicity.
Sema K1-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in animals (e.g. gene therapy, transgenics, etc.), etc. Binding assays encompass any assay where the molecular interaction of an sema K1 polypeptide with a binding, target is evaluated. The binding target may be a natural extracellular binding target such as a nerve or immune cell surface protein; or non-natural binding target such a specific immune protein such as an antibody, or an sema K1 specific agent such as those identified in screening assays such as described below. Sema K1-binding specificity may be assayed by binding equilibrium constants (usually at least about 107 Mxe2x88x921, preferably at least about 108 Mxe2x88x921, more preferably at least about 109 Mxe2x88x921), by growth cone collapse assays, by the ability to elicit sema K1 specific antibody in a heterologous host (e.g. a rodent or rabbit), etc.
For example, deletion mutagenesis is used to define functional sema K1 domains which specifically bind nerve or immune cell surface proteins in cell-based assays described below.
In a particular embodiment, the subject domains provide sema K1-specific antigens and/or immunogens, especially when coupled to carrier proteins. For example, peptides corresponding to sema K1-and human sema K1-specific domains are covalently coupled to keyhole limpet antigen (KLH) and the conjugate is emulsified in Freunds complete adjuvant. Laboratory rabbits are immunized according to conventional protocol and bled. The presence of sema K1-specific antibodies is assayed by solid phase immunosorbant assays using immobilized sema K1 polypeptides of SEQ ID NOS2, see, e.g. Table 2.
The claimed sema K1 polypeptides are isolated or pure: an xe2x80x9cisolatedxe2x80x9d polypeptide is unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, and more preferably at least about 5% by weight of the total polypeptide in a given sample and a pure polypeptide constitutes at least about 90%, and preferably at least about 99% by weight of the total polypeptide in a given sample. The sema K1 polypeptides and polypeptide domains ma! be synthesized, produced by recombinant technology, or purified from mammalian, preferably human cells. A wide variety of molecular and biochemical methods are available for biochemical synthesis, molecular expression and purification of the subject compositions, see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY) or that are otherwise known in the art.
The invention provides binding agents specific to sema K1 polypeptides, preferably the claimed sema K1 polypeptides, including agonists, antagonists, natural cell surface receptor binding targets, etc., methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. For example, specific binding agents are useful in a variety of diagnostic and therapeutic applications, especially where disease or disease prognosis is associated with improper utilization of a pathway involving the subject proteins. Novel sema K1-specific binding agents include sema K1-specific receptors, such as somatically recombined polypeptide receptors like specific antibodies or T-cell antigen receptors (see, e.g. Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory) and other natural binding agents such as Sema K1 cell surface receptors, non-natural intracellular binding agents identified in screens of chemical libraries such as described below etc. Agents of particular interest modulate sema K1 function, e.g. sema K1-modulatable cellular physiology, e.g. guidance.
Accordingly, the invention provides methods for modulating cell function comprising the step of modulating sema K1 activity, e.g. by contacting the cell with a sema K1 polypeptide, a sema K1 inhibitor, e.g. inhibitory sema K1 deletion mutants, sema K1-specific antibodies, etc. (supra). The target cell may reside in culture or in situ, i.e. within the natural host. The modulator may be provided in any convenient way, including by (i) intracellular expression from a recombinant nucleic acid or (ii) exogenous contacting of the cell. For many in situ applications, the compositions are added to a retained physiological fluid such as blood or synovial fluid. For CNS administration, a variety of techniques are available for promoting transfer of the therapeutic across the blood brain barrier including disruption by surgery or injection, drugs which transiently open adhesion contact between CNS vasculature endothelial cells, and compounds which facilitate translocation through such cells. Sema K1 polypeptides or polypeptide modulators may also be amenable to direct injection or infusion, topical, intratracheal/nasal administration e.g. through aerosol, intraocularly, or within/on implants e.g. fibers e.g. collagen, osmotic pumps, grafts comprising appropriately transformed cells, etc. A particular method of administration involves coating, embedding or derivatizing fibers, such as collagen fibers, protein polymers, etc. with therapeutic proteins. Other useful approaches are described in Otto et al. (1989) J Neuroscience Research 22, 83-91 and Otto and Unsicker (1990) J Neuroscience 10, 1912-1921. Generally, the amount administered will be empirically determined, typically in the range of about 10 to 1000 xcexcg/kg of the recipient and the concentration will generally be in the range of about 50 to 500 xcexcg/ml in the dose administered. Other additives may be included, such as stabilizers, bactericides, etc. will be present in conventional amounts. For diagnostic uses, the modulators or other sema K1 binding agents are frequently labeled, such as with fluorescent, radioactive, chemiluminescent, or other easily detectable molecules, either conjugated directly to the binding agent or conjugated to a probe specific for the binding agent.
The amino acid sequences of the disclosed sema K1 polypeptides are used to back-translate sema K1 polypeptide-encoding nucleic acids optimized for selected expression systems (Holler et al. (1993) Gene 136, 323-328; Martin et al. (1995) Gene 154, 150-166) or used to generate degenerate oligonucleotide primers and probes for use in the isolation of natural sema K1-encoding nucleic acid sequences (xe2x80x9cGCGxe2x80x9d software, Genetics Computer Group, Inc, Madison Wis). Sema K1-encoding nucleic acids used in sema K1-expression vectors and incorporated into recombinant host cells, e. for expression and screening, transgenic animals, e.g. for functional studies such as the efficacy of candidate drugs for disease associated with sema K1-modulated cell function, etc.
The invention also provides nucleic acid hybridization probes and replication/amplification primers having a sema K1 cDNA specific sequence comprising a strand of least one of: (a) SEQ ID NO:1, (b) at least 300 contiguous nucleotides of SEQ ID NO:1, (c) at least 102 contiguous nucleotides of SEQ ID NO:1, nucleotides 1017-2498, and (d) at least 36 contiguous nucleotides of SEQ ID NO:1, nucleotides 1441-2498, and sufficient to specifically hybridize with a second nucleic acid comprising the complementary strand of SEQ ID NO:1. Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5xc3x97SSPE (0.18 M NaCl, 0.01 M NaPO4, pH7.7, 0.001 M EDTA) buffer at a temperature of 42xc2x0 C. and remaining bound when subject to washing at 42xc2x0 C. with 0.2xc3x97SSPE; preferably hybridizing in a buffer comprising 50% formamide in 5xc3x97SSPE buffer at a temperature of 42xc2x0 C. and remaining bound when subject to washing at 42xc2x0 C. with 0.2xc3x97SSPE buffer at 42xc2x0 C.
The subject nucleic acids are of synthetic/non-natural sequences and/or are isolated, i.e. unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, preferably at least about 5% by weight of total nucleic acid present in a given fraction, and usually recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other than that which it is joined to on a natural chromosome. Recombinant nucleic acids comprising the nucleotide sequence of SEQ ID NO:1, or requisite fragments thereof, contain such sequence or fragment at a terminus, immediately flanked by (i.e. contiguous with) a sequence other than that which it is joined to on a natural chromosome, or flanked by a native flanking region fewer than 10 kb, preferably fewer than 2 kb, which is at a terminus or is immediately flanked by a sequence other than that which it is joined to on a natural chromosome. While the nucleic acids are usually RNA or DNA, it is often advantageous to use nucleic acids comprising other bases or nucleotide analogs to provide modified stability, etc.
The subject nucleic acids find a wide variety of applications including use as translatable transcripts, hybridization probes, PCR primers, diagnostic nucleic acids, etc.; use in detecting the presence of sema K1 genes and gene transcripts and in detecting or amplifying nucleic acids encoding additional sema K1 homologs and structural analogs. In diagnosis, sema K1 hybridization probes find use in identifying wild-type and mutant sema K1 alleles in clinical and laboratory samples. Mutant alleles are used to generate allele-specific oligonucleotide (ASO) probes for high-throughput clinical diagnoses. In therapy, therapeutic sema K1 nucleic acids are used to modulate cellular expression, concentration or availability of active sema K1.
The invention provides efficient methods of identifying agents, compounds or lead compounds for agents active at the level of a sema K1 modulatable cellular function. Generally, these screening methods involve assaying for compounds which modulate sema K1 interaction with a natural sema K1 binding target. A wide variety of assays for binding agents are provided including labeled in vitro protein-protein binding assays, immunoassays, cell based assays, etc. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds. Identified reagents find use in the pharmaceutical industries for animal and human trials; for example, the reagents may be derivatized and rescreened in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.
The following experimental sections/examples are offered by way of illustration and not by way of limitation.